Insects have low pressure circulatory systems and require diuretic hormones (DH) to control rate of urine production by Malpighian tubules. There are 13 similar DH related to CRF, but with far more diverse structures than the CRF superfamily. It has become apparent during this project that many species have two DH which are related but belong to sub-families of paralogues. We plan to continue investigating to continue investigating what appears to be two members of this family of DH in the disease vector Rhodnius prolixus. The kinins are another family of small peptides that control diuresis. They share rather conserved C-terminal pentapeptide motif and have been found to have synergistic effects with the CRF-like DH in several species of insect. We propose to identify kinins from R. prolixus and Manduca sexta; these insects represent species with disparate dietary habits, the former being an obligate blood feeder and the latter being phytophagous. We plan to study the interactions of these peptides on Malpighian tubules of the two species and their molecular mode of action. The kinins are another family of small peptides that control diuresis. They share a rather conserved C-terminal pentapeptide motif and have been found to synergistic effects with the CRF-like DH in several species of insect. We propose to identify kinins from R. prolixus and Manduca sexta; these insects represent species with disparate dietary habits, the former being an obligate blood feeder and the latter being phytophagous. We plan to study the interaction of this obligate blood feeder and the latter being phytophagous. We plan to study the interactions of these peptides on Malpighian tubules of the two species and their molecular mode of action. During the prior grant period a particularly novel DH was identified from a cockroach, Diploptera punctata, along with a CRF-like DH. This new DH proved to be structurally and functionally related to calcitonin in vertebrates. The peptide (Dippu-DH31) and the CRF-like DH from the same species have strongly synergistic effects. Due to the high potency of this peptide on related species have strongly synergistic effects. Due to the high potency of this peptide on related species of insects, it may well constitute an additional important family of DH. The existence of homologues of this DH will be investigated in other species, including R. prolixus. Studies of the effects of such peptides on the target tissue will be pursued at the molecular level. It is probable that M. sexta has at least 2 receptors for its 2CRF-like DH; one receptor has been identified and efforts to characterize other receptors will be pursued. The ligand binding site on the identified receptor will be approached by protein cross-linking and molecular biological approaches.